We have shown that the Plasmodium falciparum Pfs47 gene is critical for malaria transmission. Parasites that express Pfs47 (NF54 WT) evade mosquito immunity and survive, whereas Pfs47 knockouts (KO) are efficiently eliminated by the complement-like system. We used two alternative approaches to investigate the mechanism of action of Pfs47 on immune evasion. First, we examined whether Pfs47 affected signal transduction pathways mediating mosquito immune responses. We found that the Jun-N-terminal kinase (JNK) pathway is a key mediator of Anopheles gambiae antiplasmodial responses to P. falciparum infection and that Pfs47 allows the parasite to avoid detection by disrupting JNK signaling. Second, we used microarrays to compare the global transcriptional responses of A. gambiae midguts to infection with WT and KO parasites. The presence of Pfs47 results in broad and profound changes in gene expression in response to infection that are already evident 12 h post-feeding, but become most prominent at 26 h postfeeding, the time when ookinetes invade the mosquito midgut. Silencing of 15 differentially expressed candidate genes identified caspase-S2 as a key effector of Plasmodium elimination in parasites lacking Pfs47. We provide experimental evidence that JNK pathway regulates activation of caspases in Plasmodium-invaded midgut cells, and that caspase activation is required to trigger midgut epithelial nitration. Pfs47 alters the cell death pathway of invaded midgut cells by disrupting JNK signaling and prevents the activation of several caspases, resulting in an ineffective nitration response that makes the parasite undetectable by the mosquito complement-like system. This work was published in PNAS We identified 42 Pfs47 haplotypes based on the predicted full-length protein of 364 different isolates. The majority of African haplotypes (30/34) never migrated to another continent, while two different minor haplotypes are the most frequent ones circulating in Asia and the Americas, respectively. We found exceptionally high fixation indices (Fst) between populations from different continents. We explored the hypothesis that parasites are selected as they adapt to mosquitoes that are evolutionarily distant from those vectors present in Africa. We discovered that mosquito vectors from Africa (A. gambiae), Asia (A. dirus) and the Americas (A. albimanus) are highly susceptible to infection with P. falciparum isolates from the same region, but greatly limit infection of isolates from a different continent. Furthermore, we demonstrated that the differences in compatibility are determined by the mosquito immune system. The lines selected for the compatibility experiments express different Pfs47 haplotypes that are common in their continents of origin, but the lines also differ in their genetic background. We tested the role of Pfs47 in vector-parasite compatibility by generating transgenic lines with the same genetic background (NF54) that only differed in the Pfs47 haplotype they express, and evaluating their ability to infect different mosquito species. We show that replacement of the Pfs47 haplotype in a P. falciparum isolate is sufficient to change its compatibility to different mosquito vectors. Moreover, when a parasite expresses a compatible Pfs47 haplotype, it is no longer detected by the mosquito immune system and survives. We conclude that the mosquito immune system is a major evolutionary force that continuously selects the parasites circulating in a given region. This has important implications for the epidemiology of malaria. This work has been submitted for publication. Pfs47 allows some African P. falciparum parasites (NF54 and GB4) to evade the immune system of a highly refractory mosquito strain (L3-5), while parasites from South America (7G8) are detected and eliminated. The predicted Pfs47 proteins from these strains only differ by four amino acids. We complemented the Pfs47KO line with Pfs47 variants in which a single amino acid in the African sequence was changed to that of the South American strain. We found that changing any individual amino acid results in parasite recognition and eliminations by the immune system, indicating that there is a highly specific interaction between Pfs47 and some receptor in the mosquito. We have also generated recombinant P. falciparum parasites that express heterologous Pfs47 genes. We are testing the possibility of introducing Pfs47 orthologs from P. vivax and P. falciparum from gorillas into P. falciparum NF54 parasites as an experimental model study their interactions with the immune system of different anopheline mosquitoes and to test the potential of anti-P47 antibodies to block malaria transmission. We also produced recombinant Pfs47 in E. coli and developed a refolding protocol. The protein is immunogenic and mouse polyclonal antibodies recognize Pfs47 in the membrane of female gametocytes. We are in the process of generating monoclonal antibodies in collaboration with David Narum from Patrick Duffys section. Transmission blocking assays are currently underway using polyclonal and monoclonal antibodies. Exposure of Anopheles gambiae mosquitoes to Plasmodium infection enhances the ability of their immune system to respond to subsequent infections. However, the molecular mechanism that allows the insect innate immune system to 'remember' a previous encounter with a pathogen had not been established. In previous studies we found that challenged mosquitoes constitutively release a soluble hemocyte differentiation factor (HDF) into their haemolymph that, when transferred into Naive mosquitoes, also induces an increase in the number of circulating granulocytes (macrophage-like cells in mosquitoes) and enhances antiplasmodial immunity. In collaboration with Drs. Jose Ribeiro and Charles Serhan, we used a classical purification strategy to identify the biochemical nature of this factor and found that it consists of a Lipoxin/Lipocalin complex. We demonstrated that innate immune priming in mosquitoes involves a persistent increase in expression of Evokin (a lipid carrier of the lipocalin family), and in their ability to convert arachidonic acid to lipoxins, predominantly Lipoxin A4. Plasmodium ookinete midgut invasion triggers immune priming by inducing the release of a mosquito lipoxin/lipocalin complex. This work was published in Nature Communications. Hemocyte-specific transcripts are found in midgut samples of Plasmodium-infected mosquitoes and the transcript levels are greatly elevated when challenged mosquitoes are infected a second time. We also know that hemocytes are key players of the priming response. However, it is not clear how they enhance antiplasmodial immunity. To address this question, we labeled hemocytes in vivo with a lipophilic dye and analyzed the cells associated with the midgut. We were unable to find any intact hemoctyes. Instead, we found fluorescence-labeled microvesicles associated with Plasmodium-invaded midgut cells that were not present in uninfected controls. This indicates that hemocytes released vesicles that fuse with invaded midgut cells. We also foud HDF injection increases the number of vesicles, while pre-injection of sephadex beads that are phagocytized by hemocytes prevents vesicle release and increases parasite survival. We know from previous studies that overactivation of the Toll pathway in hemocytes, by silencing the suppressor cactus, enhances antiplasmodial immunity. We recently found that this treatment also greatly enhances the number of midgut-associated vesicles. We are investigating if preventing vesicle release, by pre-injection of beads, affects midgut epithelial responses or activation of the mosquito complement-like system.